Reprogramming an RNA-guided archaeal TnpB endonuclease for genome editing

Abstract RNA-guided endonucleases developed for genome editing have been identified exclusively from bacteria until now. Recently, the RNA-guided TnpB endonucleases encoded by bacterial IS200/IS605 and IS607 families have been reprogrammed for genome editing. In fact, these insertion sequence families are also widely distributed in archaea. However, whether archaea-derived TnpB could be used for genome editing remains unclear. Here, we found that TnpB derived from Sulfolobus islandicus REY15A (SisTnpB1) was an Mg 2+ /Mn 2+ -dependent RNA-guided endonuclease, and it could cleave DNA next to the 5’ TTTAA transposon-associated motif (TAM) to generate double-stranded DNA breaks at the 3’-end. We also found that SisTnpB1 was active at 37-85°C and tolerant to variations of TAM. Moreover, SisTnpB1 could be reprogrammed to edit bacterial cells with an editing efficiency of 100% at 37°C or 45°C. In summary, our finding expanded the resources of RNA-guided endonucleases for genome editing, especially those derived from archaea, the third domain of life.